1. DAVID MCFALL GRADE 11 CENTRAL CATHOLIC HIGH SCHOOL PJAS 2012 Sweetener Effects on Cancer Cell Proliferation

2. An Overview of Cancer Cells Cancer cells are cells that grow and divide at an irregular, unregulated pace. Apoptosis does not occur in cancerous cells; their mutations are passed on to the second generation, eventually clustering and forming tumors. Tumors can be malignant (aggressive) or benign.

3. MG63 Cancer Cell Line Human cancer cell line Osteosarcoma cells, an aggressive form of bone cancer Useful model to test the effects of variables on cancer cell proliferation

4. An Overview of Stem Cells Unspecialized cells capable of renewing themselves through cell division. Under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions. Stem cells offer new potentials for treating diseases such as diabetes and heart disease.

5. C2C12 Stem Cell Line Subclone of the mus musculus (mouse) myoblast cell line. A model type of stem cell line that was discovered in 1977 through experimentation of murine thigh muscle growth after a crush injury. Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins.

6. Variable: Sucralose Sucralose is chlorinated sucrose, replacing three hydroxyl groups with three chlorine atoms. Sucralose is met with the best reputation among artificial sweeteners with consumers. Some studies still show harmful effects of sucralose.  A study in Japan found that sucralose had some genotoxic effects; sucralose induced DNA damage to internal organs

7. Purpose To determine the effect of sucralose exposure on cancer cell and stem cell proliferation, as well as stem cell differentiation.

8. Null Hypothesis Alternative Hypothesis Sucralose WILL NOT have an effect on cancer and stem cell proliferation and stem cell differentiation Sucralose WILL significantly effect the proliferation and survivorship of cancer and stem cells and the differentiation of stem cells. Hypothesis

9. Materials Cryotank Two 75mm 2 tissue culture treated flasks Twelve 25 mm 2 tissue culture treated flasks Fetal bovine serum (FBS) C2C12 Myoblastic Stem Cell Line MG63 Osteosarcoma Cancer Cell Line Trypsin-EDTA Pen/strep Macropipette + sterile macropipette tips (1 mL, 5 mL, 10, mL, 20 mL) Micropipettes + sterile tips DMEM Serum - 1% and Complete Media (4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate + [ 10% fetal bovine serum for complete]) Equal Sucralose (powder) 75 mL culture flask Incubator Nikon Inverted Microscope 24 well plate Laminar Flow Hood Laminar Flow Hood UV Sterilizing Lamp Labeling Tape Hemocytometer Sterile PBS Ethanol (70% and 100%) Purple Nitrile gloves

10. Procedure: Cell Culturing A 1 mL aliquot of C2C12 cells and MG63 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in two 75mm 2 culture flask yielding a cell density of approximately 10 6 to 2x10 6 cells. The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO 2 ) for 2 days until a cell density of approximately 4x10 6 to 5x10 6 cells/mL was reached. The culture was passed into two sets of 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO 2.

11. Procedure: Proliferation Experiment- Day 0 (Addition of Variable) After trypsinization, cells from all of the flasks were pooled into 1 common 75mm 2 flask (cell density of approximately 1 million cells/mL). 0.1 mL of the cell suspension was added to six 25 mm 2 tissue culture treated flasks containing 5 mL of DMEM (com) media, creating a cell density of approximately 10 5 cells per flask. The stock solution of sucralose (10 -3 ) was created using 5 mL of ethanol and a 1 gram packet of sweetener (containing 11 mg of the active ingredient, sucralose). This was then used in a serial dilution to create a 10 -4 concentration. The following concentrations of variable (next page) were added to the flasks. 6 flasks for each cell line (two for each concentration of variable). The cells were incubated at 37°C, 5% CO 2 for the remainder of the study.

12. Proliferation experiment: Differentiation Experiment : 0X10x Stock0 mL5 µL50 µL Media5 mL4.995 mL4.950 mL Total5 mL 0X10x Stock0 mL1 µL10 µL Media1 mL0.999 mL0.990 mL Total1 mL Concentrations of Variable

13. Procedure: Proliferation Experiment- Days 1 and 3 Day 1  Using one flask from each group, cell densities were determined as follows:  The cells were trypsinized and collected into cell suspension.  25 µl aliquots were transferred to a Hemocytometer for quantification (eight counts per flask). Day 1 and Day 3  The previous procedure for determining densities was used again, and a Nikon Inverted Microscope was used to take images of representative areas of each flask.

14. Procedure: Differentiation Experiment Day 1 The original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation. Day 1, 3, 5, and 7 Using the Nikon Inverted Microscope, images of representative areas of each of the groups were taken.

15. Results of Proliferation Analysis (C2C12)

16. Results of Proliferation Analysis (MG63)

17. Dunnett’s Test Results ConcentrationT-ValueT-Critical (0.05) Variation C2C12--- X0.310 3.03 Insignificant 10X5.092 3.03 Significant MG63--- X4.648 3.03 Significant 10X1.716 3.03 Insignificant

18. Differentiation Results: Day 1 Control X10X

19. Differentiation Results: Day 7 Control X 10X

20. Conclusions Proliferation  C2C12  Based upon the results gathered from the ANOVA and Dunnett’s statistical analyses, it appears that the addition of sucralose in high concentration significantly affected stem cell proliferation.  MG63  Based upon the results gathered from the ANOVA and Dunnett’s statistical analyses, it appears that the addition of sucralose in low concentration significantly affected cancer cell proliferation. Differentiation  It appears as if sucralose does not affect myotube differentiation

21. Limitations Extensions Hemocytometer counts in proliferation experiment were qualitative  Cell staining could quantify the cell counts. Differentiation test was qualitative Obtain an LD-50 for sucralose Use a wider range of concentrations Test synergistic effects of sweeteners Future Changes

22. Works Cited Mark Krotec, PTEI Merisant Company http://www.ncbi.nlm.nih.gov/pubmed /12160896 http://www.ncbi.nlm.nih.gov/pubmed /12160896 http://cancerres.aacrjournals.org/cont ent/40/3/734.full.pdf http://cancerres.aacrjournals.org/cont ent/40/3/734.full.pdf

23. Statistical Analyses of the Proliferation Results ANOVA  Compares variation within groups to variation between groups.  Using the ANOVA, a p-value less than the alpha of.05 was gathered (significant variation).  Reject the null hypothesis. Dunnett’s test  Compares each experimental group to control individually.  0.05 alpha was used, and the t-value compared to the t- critical value of 3.03